Ninjurin1 was first reported by Toshiyuki Araki et al in 1996 (Araki, T. & Milbrandt, J., Neuron 17, 353-361, 1996). It was found during the screening of a gene up-regulated in Schwann cells after damage caused by transection or crush in sciatic nerve.
Phylogenetic tree was made with proteins having homology with Ninjurin based on protein information in GeneBank (Zhang, S. et al., Genes & development 20, 1899-1910, 2006). It was reported that vertebrate have two Ninjurin proteins, Ninjurin1 and Ninjurin2. In fact, Ninjurin1 and Ninjurin2 were found in vertebrate such as human, mouse and rat. Ninjurin identified in invertebrate is classified into three types, A, B, and C, and specifically identified in drosophila, mosquito, and so on. Human Ninjurin1 shows 90% homology with mouse Ninjurin1 (Chadwick, B. P. et al., Genomics 47, 58-63, 1998). In the meantime, human Ninjurin1 shows 55% homology with human Ninjurin2 (Araki, T. & Milbrandt, J., J Neurosci 20, 187-195, 2000).
Human Ninjurin1 is located at chromosome 9q22 and is composed of 152 amino acids. Mouse Ninjurin1, in the meantime, is located at chromosome 13 and is composed of 152 amino acids. Two transmembrane domains are predicted in the amino acid sequence of Ninjurin1. It was also confirmed by experiments that Ninjurin1 is the protein located in cell membrane (Araki, T. & Milbrandt, J., Neuron 17, 353-361, 1996). Accordingly, it can be predicted that N-terminal region of Ninjurin1 is stretched long out of cell.
Ninjurin1 is expressed in diverse tissues. For example, it is expressed at RNA level in the heart, brain, placenta, lung, liver, SK. Muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small int., colon, blood, adrenal gland and dorsal root ganglia (DRG). It is expressed at protein level in the liver, kidney, thymus, uterus, adrenal gland, retina and dorsal root ganglia (Araki, T. et al., The Journal of biological chemistry 272, 21373-21380, 1997).
The functions of Ninjurin1 known so far are in relation to 1) cell adhesion, 2) neurite outgrowth, 3) cellular senescence, and 4) cancer.
Particularly, regarding the function of Ninjurin1 in relation to cell adhesion 1), it was reported via cell aggregation experiment performed with Jurkat T cell leukemia that Ninjurin1 increased aggregation among cells (Araki, T. & Milbrandt, J., Neuron 17, 353-361, 1996). For the cell adhesion, polymerization of actin filaments, oxidative phosphorylation, divalent cation and proper pH (pH 7-11) are required (Araki, T. et al., The Journal of biological chemistry 272, 21373-21380, 1997). In the study using drosophila, Ninjurin A protein digested with MMP1 (matrix metalloproteinase 1) acted as a signal molecule to inhibit cell adhesion (Zhang, S. et al., Genes & development 20, 1899-1910, 2006).
To investigate the function of Ninjurin1 in relation to neurite outgrowth 2), CHO cells were monolayer-cultured, on which DRG neurite cells were cultured. When CHO cells over-expressing Ninjurin1 were used, neurite cell proliferation was increased (Araki, T. & Milbrandt, J., Neuron 17, 353-361, 1996). When the amino acid sequence ranging from the 26th to the 37th residue of Ninjurin1 protein was modified, neurite cell proliferation was inhibited (Zhang, S. et al., Genes & development 20, 1899-1910, 2006). When DRG neurite cells and skin-derived fibroblast-like cells (FLCs) were co-cultured, when Ninjurin1 was expressed, neurite cells were being proliferated but when Ninjurin1 was inhibited by an antibody not to be functioning, neurite cell proliferation was inhibited (Jerregard, H. et al., Journal of neurocytology 30, 327-336, 2001).
Regarding cellular senescence 3), when Ninjurin1 was over-expressed, cell cycle was arrested in G1 stage after p21WAF1/Cip1 transcription, resulting in a significant inhibition of cell proliferation. Besides, when Ninjurin1 was over-expressed, senescence-associated β-galactosidase activity and autofluorescence pigment were increased. Ninjurin1 is also up-regulated in hepatocellular carcinoma tissue, suggesting that Ninjurin1 might be involved in cellular senescence which is the target of anti-cancer treatment (Toyama, T. et al., Journal of hepatology 41, 637-643, 2004).
In studies of Ninjurin1 in relation to cancer 4), Ninjurin1 was confirmed to be up-regulated in hepatocellular carcinoma including virus infection in the liver or cirrhosis (Kim, J. W. et al., Molecules and cells 11, 151-157, 2001). Ninjurin1 was also increased in acute lymphocytic leukemia. Ninjurin1 was directly increased during the screening of a gene regulated by the tumor suppressing protein p53 using microarray (Kannan, K. et al., Oncogene 20, 2225-2234, 2001).
However, it has not been disclosed yet whether Ninjurin1 is involved in the functions of macrophages, vascular decrease and inflammation induction.
So, the present inventors have been studied on the involvement and mechanism of Ninjurin1 in relation to macrophages, during which the inventors confirmed that Ninjurin1 was expressed specifically in macrophages around blood vessels, increased cell-matrix and cell-cell adhesion, increased Wnt7b (Wingless-type MMTV integration site family, member 7B) and Ang2 (angiopoietin-2) expressions, and accelerated apoptosis of vascular endothelial cells (VECs) by reducing Ang1 (angiopoietin-1) expression. Further, the present inventors completed this invention by confirming that Ninjurin1 was up-regulated when inflammation was induced by LPS in vivo and in vitro and increased iNOS expression and NO generation and accordingly confirming that Ninjurin1 increased the activity of macrophages to induce inflammation.